Antimicrobial peptides as models for the evolution of gene duplication Antimicrobial peptides (AMPs) are a vital part of the humoral immune response for eukaryotes. Gene family expansion through duplication has long been recognized as a means of generating evolutionary novelty though the evolutionary processes leading from gene duplication to novel function is not well established. One striking characteristic of the evolution of AMPs is the high rate of gene duplication in AMP gene families. The proposed research will use duplications in AMP gene families to better understand both AMP function and the evolution of gene duplication. Using AMPs to study gene duplication may have applicability to human health and provides built-in replication of gene duplications across families in Drosophila. Furthermore, the ability to perform high throughput experiments and genetic manipulations in Drosophila and the existence of a set of expected phenotypes involved in AMP function make the system tractable. Aim 1 is a comprehensive analysis of AMP evolution within and between species to address the role that selection plays in evolution after gene duplication and how gene expression diverges with between species and paralogs. Part of this analysis will examine expression of these AMPs on a very fine scale both spatially (across tissues) and temporally. The training component of Aim 1 includes state-of-the art population genetic analyses as well as fine scale gene expression studies. Aim 2 consists addresses the function of specific AMP paralogs in response to both oral infection and systemic infection using engineered gene knockouts and employing a large variety of pathogens since we have evidence that AMP immune response is pathogen-specific. Based on the results from this experiment, we will create transgenic lines with multiple copies of a particular AMP to test the effects of gene dosage, perform promoter swapping experiments between paralogs to test how changes in regulatory sequence might influence expression en route to subfunctionalization. Training in Aim 2 consists of the construction of transgenic lines using developing genome editing technologies to address these questions. Aim 3 will focus specifically on AMP duplicates segregating in natural populations to address the nature of paralog function shortly after the initial duplication event. We will employ the same basic framework as in Aims 1 and 2 examining patterns of variability, gene expression and paralog function but focusing on these segregating tandem duplicates. We will additionally create transgenic tandem duplicates to perform functional assays controlling for background effects. Because of the replicated nature of AMP gene duplicates across gene families, we expect to draw conclusions about the evolution of gene duplication. The training portion of the proposed research will complement the applicant's previous experience and position him for a productive research career. Cornell University and the Lazzaro and Clark labs together have the resources and expertise to ensure the successful completion of the training phase of the grant.